A student has asked for some clarification - I'll do my best to help!
(The questions were probably a midnight brain-dump many months ago)
[... from a student's query...]
> In question number 7, what means "Are there more reported in PDB?"? Maybe
> whether are the macromolecules in the PDB file under examination present
> also in other entries?
The point is that many PDB files contain several molecules. This can be
because:
- the protein consists of more than one component (hetero or
homo-oligomer)
- there are some small molecules (substrate, solvent, etc.)
- the crystal structure has more than one molecule in the asymmetric
unit.
In all of these cases if you display the molecule using rasMol you might
get a surprise! "I wasn't expecting THAT!"
> I did not understand well the meaning of questions 9 and 10.
9.
Many (?most) proteins crystallise with single molecules in the
asymmetric unit - an example is myoglobin (2MBD from memory). Sometimes
there are two or more molecules packed tightly together which MAY or MAY
NOT be biologically relevant! A lot of experimental work is often done
precisely to find whether a molecule acts as monomers or oligomers.
Sometimes the evidence is conflicting.
Biological reasons for oligomerisation are allostery (where
an enzyme (for example) dimerises and there are two binding sites. When
a ligand binds to one it affects the molecular conformation and hence the
activity and/or the ability of more molecules to bind. A classic case is
haemoglobin, where the binding of one O2 affects the other sites.
Another reason for dimerisation is to bring molecules together and
trigger an effect. This is strongly argued for growth factor receptors
(e.g. EGF. insulin,)
Some molecules aggregate in larger numbers. This is particulalry
true for proteins with a structural role such as viral capsids. So you
might find 60 copies of your protein in an icosaherdal virus or 34 in the
disk of TMV.
And some molecules are infinite polymers (e.g. DNA, TMV, etc.) You
probably don't have these in your exercise, but....
10.Is there any evidence **in the crystal** that the protein is not
homogeneous?
Although crystallisation is a good method of purifying proteins, it's
often possible for co-crystallisation of similar molecules. So, if you
have a molecule which is processed (e.g. it might or might not have a
N-terminal Met) both can crystallise in the same structure. In this case
the denisty round the variable site will be poorly defined and the
authors may comment on this (e.g. 'MET 1 appears to be only partly
present and the density is poor', or: 'The active site binds
alkyl sulphonyl fluoride and about 50% of the molecules appear to have
this covalent attachment'...
> In question 12 what means "Were all atoms located? (Roughly)"?
If parts of the structure are poorly defined it may be difficult to
locate them in the X-ray map. See the discussion 2-3 weeks ago about
pyroglutamate at the N-terminus. A strong clue is if the SEQRES cards
mention residues which aren't in the ATOM cards.
["(Roughly)" ... I think I meant don't worry too much about every atom
:-)]
> Thank you very much and happy Easter,
> best regards
.....
Thanks you :-) I enjoy this.
P.
Peter Murray-Rust 44-181-9662554 T "Nothing exists except atoms and empty
pmr1716@ggr.co.uk 44-181-9662109 F space; all else is opinion" Democritos.
Biomolecular Structure, Glaxo Wellcome, Greenford, Middx, UB6 0HE, UK
& p.murray-rust@mail.cryst.bbk.ac.uk :http://www.cryst.bbk.ac.uk/PPS/index.html